Single Cell analysis using AtomicForce Microscopy (AFM)

Replication of biological cells for the purpose of imaging and analysis under electron and scanning probe microscopy has facilitated the opportunity to study and examine some molecular processes and structures of living cells in a manner that were not possible before. The difficulties faced in direct cellular analysis when using and operating Atomic Force Microscopy (AFM) in situ for morphological studies of biological cells have led to the development of a novel method for biological cell studies based on nanoimprint lithography. The realization of the full potential of high resolution AFM imaging has revealed some very important biological events such as exocytosis and endocytosis. In this work, a soft lithography Bioimprint replication technique, which involved simple fabrication steps, was used to form a hard replica of the cell employing a newly developed biocompatible polymer that has fast curing time at room temperature essential for this process. The structure and topography of the rat muscle cell and the endometrial (Ishikawa) cancer cell were investigated in this study. Cells were cultured and incubated in accordance with standard biological culturing procedures and protocols approved by the Human Ethics Committee, University of Otago. An impression of the cell profile was created by applying a layer of the polymer onto the cells attached to a substrate and rapidly cured under UV-light. Fast UV radiation helps to lock cellular processes within seconds after exposure and replicas of the cancer cells exhibit ultra-cellular structures and features down to nanometer scale. Elimination of the AFM tip damping effects due to probing of the soft biological tissue allows imaging with unprecedented resolution. Highxx resolution AFM imagery provides the opportunity to examine the structure and topography of the cells closely so that any abnormalities can be identified. Craters that resemble granules and features down to 100 nm were observed. These represent steps on a transitional series of sequential structures that indicate either an endocytotic or exocytotic processes, which were evident on the replicas. These events, together with exocytosis, play a very significant part in the tumorigenesis of these cancer cells. By forming cell replica impressions, not only have they the potential to understand biological cell conditions, but may also benefit in synthesizing three dimensional (3-D) scaffolds for natural growth of biological cells and providing an improvement over standard cell growth conditions. Further examinations by observing the characteristic behaviour of the plasma membrane when the cells were induced by certain compound such as cobalt chloride (CoCl2) under control and stimulated conditions have brought in the opportunity to examine the effect of this stimulant in inducing apoptosis in many different kinds of cells. Numbers of pores formed on the cells membrane were found to increase significantly after the cells where induced with CoCl2 that correlated well with the level of vascular endothelial growth factor (VEGF) receptors expression, which contributed to tumour growth. This indicates CoCl2 has exaggerated the expression of the VEGF growth factor. Investigations were also done to the cells using functionalized nanoparticles as bio-markers to establish the connection between exocytosis with nanopores found on the membrane surfaces of the cells. These microbeads were found attached to sites surrounding the nucleus of the cell and higher numbers of visible beads would confirm that there was an up-regulation of the VEGF expression in cells induced by CoCl2. All these can contribute to expanding the knowledge about exocytosis and fundamental physiology of cells, and also assist in understanding diseases especially cancer